© 2002 Wiley-Liss, Inc.
Cytometry 48:93–96 (2002)
Use of SYTOX Green Dye in the Flow Cytometric
Analysis of Bacterial Phagocytosis
J.J. Gaforio,* M.J. Serrano, E. Ortega, I. Algarra, and G. Alvarez de Cienfuegos
Unit of Microbiology. Department of Health Sciences, Faculty of Experimental Sciences, University of Jaén, Jaén, Spain
Received 14 November 2001; Revision Received 5 February 2002; Accepted 22 March 2002
Background: Fluorescein isothiocyanate (FITC) is used
widely to label the targets used in flow cytometric phago-
cytosis assays. Unfortunately, the fluorescence intensity of
phagocytosed FITC-labeled targets is influenced by
changes in intracellular pH level, making quantitative mea-
surements with this fluorophore problematic. We de-
scribe the use of SYTOX green nucleic acid stain to mea-
sure phagocytosis by flow cytometry.
Methods: Suspensions of isopropyl alcohol-permeabilized
Escherichia coli DH5␣ were stained with the SYTOX
green dye and then incubated with resident peritoneal
macrophages. The samples were analyzed by flow cytom-
etry and phagocytosis was determined by gating the cells.
Results: Results are expressed as percentage of phago-
cyte-associated green fluorescent cells. The validity of the
A variety of methods are available for quantifying phago-
cytosis (1). Flow cytometry offers rapid and reproducible
phagocytosis measurements of single cells in suspension
(2). The targets commonly used in flow cytometric phago-
cytosis assays are bacteria, zymosan, fungi, and latex/
polystyrene beads. For this purpose, fluorescein isothio-
cyanate (FITC) is used widely to label these targets, but
the fluorescence intensity of phagocytosed FITC-labeled
targets is influenced by changes in the intracellular pH
level, which disturb the quantitation of phagocytosis (3).
Other fluorescent dyes have been used, but the efficiency
as label cells is governed by their selectivity, brightness,
excitation, and emission maxima. Recently, we described
the use of 7-amino-actinomycin D (7AAD) in the analysis
of Candida albicans phagocytosis (4). 7AAD is a fluores-
cent DNA-binding agent, previously used just for studies
on apoptotic cells (5).
In this article, we report the use of SYTOX green to
measure Escherichia coli phagocytosis by flow cytometry.
SYTOX green is a high-affinity nucleic acid stain that does
not penetrate living cells and is used to assess the integrity
of the plasma membranes of bacteria (6 – 8). It is particu-
larly useful with both gram-positive and gram-negative
bacteria, where an exceptionally bright signal is required.
After a brief incubation with SYTOX green, the nucleic
acids of dead cells fluoresce bright green (emission max-
method was shown by the effects of a phagocytosis inhib-
itor (incubation at 4°C) or enhancer (gamma interferon
[IFN-␥] treatment) being accurately assessed with this
assay.
Conclusions: The method described was reproducible
and provides an advantageous alternative to the use of
FITC to label bacteria for the flow cytometric measure-
ment of target uptake by phagocytic cells. Cytometry 48:
93–96, 2002. © 2002 Wiley-Liss, Inc.
Key terms: phagocytosis; Escherichia coli; SYTOX green;
macrophages; flow cytometry
ima at 523 nm) when excited with the 488-nm spectral
line argon ion laser. These properties, combined with its
more than 500-fold fluorescence enhancement upon nu-
cleic acid binding and high quantum yield, make SYTOX
green an ideal candidate for this fluorescence-based assay.
MATERIALS AND METHODS
Bacterial Cultures
All experiments were conducted with E. coli DH5␣. E.
coli were harvested in log phase from a 4-h culture in Luria
Bartani (LB) broth (Scharlau, Barcelona, Spain). Bacteria
were washed twice at 8,800g for 3 min and spectropho-
tometrically adjusted to a concentration of 1 ⫻ 108 bac-
teria/ml in phosphate-buffered saline (PBS; Sigma, St.
Louis, MO).
Permeabilization of Bacteria
Aliquots (1 ml) of cells were pelleted by centrifugation
at 8,800g for 3 min and resuspended in 1 ml of 70%
Grant sponsor: Plan Andaluz de Investigación; Grant number: CTS 442.
*Correspondence to: J.J. Gaforio, Department of Health Sciences, Fac-
ulty of Experimental Sciences, University of Jaén, Paraje Las Lagunillas s/n,
23071 Jaén, Spain.
E-mail: jgaforio@ujaen.es
Published online in Wiley InterScience (www.interscience.wiley.com).
DOI: 10.1002/cyto.10107

94 GAFORIO ET AL.
isopropyl alcohol for 1 h. The bacterial suspension was
subsequently pelleted by centrifugation, washed twice in
sterile 0.9% NaCl in water, and stored at 4°C until use.
SYTOX Green Staining
Stock solution of SYTOX green dye is supplied as a
5-mM solution in dimethylsulfoxide (DMSO; Molecular
Probes, Eugene, OR). Isopropyl alcohol-permeabilized E.
coli suspensions were stained (5 min at room tempera-
ture) with 5 ␮M SYTOX green. The bacterial suspension
was pelleted by centrifugation and washed twice in sterile
0.9% NaCl in water. The targets were opsonized with 50
␮l fetal calf serum (FCS; Flow Laboratories, Irvine, UK)
(non– heat-inactivated) for 10 min at 37°C in a tempera-
ture-controlled shaker. The bacterial suspension was pel-
leted by centrifugation and resuspended in 1 ml complete
medium RPMI 1640 (Sigma) supplemented with 10% heat-
inactivated FCS, 1% penicillin G/streptomycin solution
(Sigma), 1% L-glutamine (Sigma), 1% sodium pyruvate (Sig-
ma), and 1% HEPES (Flow Laboratories).
Preparation of Peritoneal Macrophage Suspensions
Six to 8-week-old male BALB/c mice were killed and
injected intraperitoneally (i.p.) with 3 ml phosphate-buff-
ered saline (PBS). The peritoneal fluids were aspirated,
washed twice by centrifugation at 200g and 4°C for 10
min, and adjusted to 5 ⫻ 106 viable cells per milliliter in
complete medium RPMI 1640.
In some experiments and with the aim of enhancing the
phagocytic capacity of peritoneal macrophages, the cells
were cocultured for 48 h with 100 IU/ml gamma inter-
feron (IFN-␥; Boehringer Mannheim, Germany) prior to
use in the phagocytosis assay. After incubation, adherent
cells were detached from the wells (24-well tissue culture
plates, Nunc, Roskilde, Denmark) by incubating the cells
in ice-cold 0.02% EDTA/PBS solution for 10 min.
Phagocytosis Assay and Flow cytometric Analysis
A flow cytometric-based assay was developed for quan-
tifying phagocytosis of bacteria stained with SYTOX green
by peritoneal cells. In a polystyrene tube (13 ⫻ 75 mm2;
Soria Greiner, Spain), 100 ␮l containing 5 ⫻ 105 resident
peritoneal macrophages were incubated with 100 ␮l con-
taining 7.5 ⫻ 106 alcohol-permeabilized stained E. coli to
start the phagocytosis reaction. The reaction resulted in a
15:1 ratio (previously determined to be optimal for detect-
ing maximal ingested and minimal adhered bacteria). The
mixture was incubated for 15 min on a shaking plateau at
37°C in a 5% CO2 incubator and the reaction was arrested
by adding 200 ␮l ice-cold isotonic-buffered paraformalde-
hyde (2%). After incubation, the cells were washed twice
in PBS (at 100g and 4°C for 10 min) to remove nonphago-
cytosed bacteria and then they were resuspended in 1 ml
of ice-cold 2% paraformaldehyde solution. Samples were
analyzed on an EPICS Elite ESP flow cytometer (Coulter,
Hialeah, FL) within 30 min of cell fixation. At least 5 ⫻ 103
events were measured for each sample. Computerized
gating in forward scatter (FSC) and side scatter (SSC) was
used to eliminate cell debris and residual free bacteria
10970320, 2002, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/cyto.10107, Wiley Online Library on [18/04/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
FIG. 1. Flow cytometric analysis of resident peritoneal macrophages
incubated at 37°C with opsonized unstained E. coli. The percentage value
represents the proportion of phagocytic cells. Mean ⫾ SD ⫽ 0.9 ⫾ 0.15
(n ⫽ 3). SYTOX green fluorescence is collected in the FL1 channel and
presented on a log scale.
from macrophages in the analysis. Scattergrams were gen-
erated by combining FSC with SYTOX green fluorescence
(FL1 log). The fluorescence histogram was divided into
two regions representing background fluorescence of
nonphagocytosing cells and a high signal for cells with
associated SYTOX green-labeled bacteria. Phagocytosis
was determined by gating the cells and was expressed as
percentage of phagocyte-associated green fluorescent
cells.
RESULTS AND DISCUSSION
The results are representative of at least three indepen-
dent experiments. We used isopropyl alcohol-permeabi-
lized E. coli DH5␣ as targets. The opsonization and phago-
cytosis times were optimal in our flow cytometric assays.
Figure 1 shows a negative control where unstained E. coli
was used. Figure 2 summarizes the results obtained when
peritoneal macrophages were incubated at 37°C with op-
sonized stained E. coli. The percentage of phagocytic cells
observed was 85%. The effects of a phagocytosis inhibitor
or enhancer were assessed with this assay and the results
are shown in Figures 3 and 4, respectively. When cells
were incubated at 4°C with opsonized stained E. coli,
phagocytosis was inhibited (12%) compared with control
cells (85%; Fig. 3). At low temperatures (4°C), cells are
unable to ingest but can still bind bacteria (9). Thus, 12%
represents phagocytic cells with adhered bacteria. On the
other hand, when the peritoneal macrophages were
cocultured with recombinant IFN-␥ at 100 IU/ml for 48 h
prior to the phagocytosis assay, 100% of the cells were
subsequently phagocytic (Fig. 4).

SYTOX GREEN DYE AND BACTERIA PHAGOCYTOSIS
FIG. 2. Flow cytometric analysis of resident peritoneal macrophages
incubated at 37°C with opsonized SYTOX green stained E. coli. The
percentage value represents the proportion of phagocytic cells. Mean ⫾
SD ⫽ 83.8 ⫾ 2.3 (n ⫽ 3). SYTOX green fluorescence is collected in the
FL1 channel and presented on a log scale.
FIG. 3. Flow cytometric analysis of resident peritoneal macrophages
incubated at 4°C with opsonized SYTOX green stained E. coli. The
percentage value represents the proportion of phagocytic cells with
adhered bacteria. Mean ⫾ SD ⫽ 11.3 ⫾ 1.0 (n ⫽ 3). SYTOX green
fluorescence is collected in the FL1 channel and presented on a log scale.
10970320, 2002, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/cyto.10107, Wiley Online Library on [18/04/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
95
FIG. 4. Flow cytometric analysis of resident peritoneal macrophages
pretreated with IFN-␥ and incubated at 37°C with opsonized SYTOX
green stained E. coli. The percentage value represents the proportion of
phagocytic cells. Mean ⫾ SD ⫽ 99.6 ⫾ 0.3 (n ⫽ 4). SYTOX green
fluorescence is collected in the FL1 channel and presented on a log scale.
A variety of fluorochromes have allowed flow cytomet-
ric measurement of phagocytosis (2,4). We report the use
of SYTOX green dye in the measurement of bacteria up-
take by phagocytic cells. It is particularly useful to label
bacteria, where an exceptionally bright signal is required
to analyze samples with flow cytometers. Unlike other
dyes, the SYTOX green nucleic acid stain shows little base
selectivity. Combined with its more than 500-fold fluores-
cence enhancement upon nucleic acid binding and high
quantum yield, SYTOX green is an ideal candidate for this
fluorescence-based assay. On the other hand, fluorescence
intensity of phagocytosed targets previously labeled with
nucleic acid dyes is less influenced by changes in the
intracellular pH than those of bacteria-labeled with FITC
(2). Like FITC, staining with the SYTOX green dye may be
used in conjunction with other fluorochromes for mul-
tiparameter analysis. The method described was repro-
ducible and provides an advantageous alternative to the
use of FITC to label bacteria for the flow cytometric
measurement of target uptake by phagocytic cells.
ACKNOWLEDGMENTS
M.J. Serrano was supported by a fellowship from the
University of Jaén.
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